Adeno-associated virus (AAV) is a leading gene delivery system currently under development. AAV serotypes are highly neurotrophic and capable of high levels of long term gene expression following a single injection in the central nervous system (CNS). AAV gene transfer in glioma (GL) models has revealed that transduction efficiencies are highly variable between serotypes, and despite reports of limited AAV transduction of microglia (MG) in vitro, transduction of these cells in animals is poor. The major goal of this proposal is to develop AAV vectors characterized by selective transduction of GL cells as well as the MG that infiltrate these tumors, and to apply these and native AAV serotypes in GL immunotherapy. In addition to targeting the GL cells themselves, the high number of MG that infiltrate GL suggests another potentially exploitable target for developing GL gene therapy. MG cells are the primary immune effectors of the CNS with demonstrated tumor killing capacity in vitro. Evidence to support a similar role in vivo comes from the finding that abundant, activated MG are a consistent feature of human and experimental GL. Yet the function of MG and other GL infiltrating lymphocytes (such as cytotoxic T cells) are inhibited within the immunosuppressive GL microenvironment. Therefore, we will test the hypotheses that (1) AAV vectors with GL and tumor-infiltrating MG selective transduction profiles can be selected for using replication-competent AAV combinatorial capsid protein libraries characterized by randomized virion surface loops;(2) AAV vectors that display dominant T-cell epitopes on the capsid surface, while simultaneously expressing the full length GL antigen, can be employed as "prime-boost" vaccine carriers for GL immunotherapy;and (3) systemic and central delivery of AAV vectors that express immune modifiers of MG and/or regulatory T-cells can elicit potent immune responses to GL. While advancing the AAV vector platform generally, the development and characterization of these vectors will provide valuable insight into the immune response to GL. Furthermore, the development of AAV vectors that selectively target MG would have broad applicability in a wide range of CNS disorders. Public Health Relevance: Glioma is the most common brain tumor in adults. Despite aggressive treatment with surgery, radiotherapy, and chemotherapy prognosis for survival is poor. Significant advances have been made in understanding anti-tumor immunity in the glioma patient This proposal evaluates the potential of adeno-associated virus gene therapy to boost the immune system's ability to mount a more effective attack on glioma tumor cells.